The goal of this proposal is to gain insight into the molecular events that lead to transformation of human B cells by Epstein-Barr virus. Specifically, we will identify cellular genes whose expression is induced by EBV and investigate their regulation and role in transformation. EBV-induced (EBVI) cellular genes will be selected from a subtraction cDNA library that I have generated from an EBV-positive B cell line (BJAB-B958) after enrichment for sequences that are differentially expressed in the EBV-positive line relative to its EBV-negative parent line (BJAB). Transcriptional expression of subtracted cDNAs will be examined in EBV-positive and -negative lines, including EBV-immortalized B cells. cDNAs that are expressed in EBV-infected cells but are not derived from viral genes will be selected for sequence analysis and additional studies of regulation and/or function. A major focus of this proposal is the elucidation of the mechanisms by which identified EBVI genes are regulated in the presence of EBV. Nuclear run-off and mRNA stability analysis will be performed to ascertain if regulation is at the transcriptional level. Mechanisms that control expression of transcriptionally-regulated EBVI gene(s) will be further explored by identifying cis-acting regulatory elements. DNA fragments containing the 5' flanking region of the EBVI gene will be obtained from a human genomic library. 5' cis-acting regulatory elements will be identified by reporter gene assays in EBV-positive and -negative BJAB cells following transfection with a series of constructs containing DNA fragments from the 5' region of the gene. Mapping of the DNAse hypersensitive sites of the gene will provide additional information about its regulatory elements. Trans-acting factors that interact with cia-acting elements of the gene will be investigated by comparison of nuclear extracts from EBV-positive and -negative cells in gel retardation assays and DNA footprinting analysis. A second focus of this proposal is the determination of the functional role of EBVI genes in transformation of B cells. The functions of EBVI genes will be studied by expressing selected EBVI genes (those that are novel and whose sequence predicts growth-regulatory or transforming properties) in EBV-negative B cell lines and normal B cells. Transfectants will be examined for altered growth properties induced by the gene.